The interaction of fungi with dendritic cells: implications for Th immunity and vaccination.

Human beings are continuously exposed to fungi, yet they rarely get fungal diseases. The delicate balance between the host and these otherwise harmless pathogens may turn into a parasitic relationship, resulting in the development of severe infections. The ability to reversibly switch between unicellular and filamentous forms, all of which can be found in infected tissues, is thought to be important for virulence. Efficient responses to the different forms of fungi require different mechanisms of immunity. Dendritic cells (DC) are uniquely able at decoding the fungus-associated information and translating it in qualitatively different T helper (Th) immune responses, in vitro and in vivo. Myeloid DC phagocytosed yeasts and hyphae of Candida albicans and conidia and hyphae of Aspergillus fumigatus, both in vitro and in vivo. Phagocytosis occurred through distinct phagocytic morphologies, involving the engagement and cooperativity of distinct recognition receptors. However, receptor engagement and cooperativity were greatly modified by opsonization. The engagement of distinct receptors translated into disparate downstream signaling events, ultimately affecting cytokine production and costimulation. In vivo studies confirmed that the choice of receptor and mode of entry of fungi into DC was responsible for Th polarization and patterns of susceptibility or resistance to infection. Adoptive transfer of different types of DC activated protective, nonprotective and regulatory T cells, ultimately affecting the outcome of infection. The conclusions are that the selective exploitation of receptors and mode of entry into DC may determine the full range of host's immune relationships with fungi and have important implications in the design of vaccine-based strategies.

Vaccination of mice against invasive aspergillosis with recombinant Aspergillus proteins and CpG oligodeoxynucleotides as adjuvants.

In a murine model of invasive pulmonary aspergillosis, dendritic cells (DCs) pulsed with Aspergillus antigens induced the activation of CD4(+) Th1 cells capable of conferring resistance to the infection. Here we show that the combined, local delivery of unmethylated CpG oligodeoxynucleotides (ODNs) and the Asp f 16 Aspergillus allergen resulted in the functional maturation and activation of airway DCs capable of inducing Th1 priming and resistance to the fungus. Therefore, ODNs act as a potent adjuvant for the vaccine-induced protection against the fungus by promoting dominant Th1 response to Aspergillus antigens and allergens.

Immunogenicity and protective effect of recombinant enolase of Candida albicans in a murine model of systemic candidiasis.

Enolase, a 46-kDa glycolytic enzyme, is an immunodominant antigen of the opportunistic pathogen Candida albicans. A recombinant 6 x His-tagged enolase was studied, in conjunction with interleukin-12 (IL-12), as an adjuvant for cytokine induction favouring protection in a murine model of haematogenous candidiasis. Mice immunized with enolase plus IL-12 showed increased antibody titres against enolase, as well as increased median survival time and decreased fungal burden in kidneys, in comparison to non-immunized or IL-12-treated mice. This increased survival was attributable to enolase-induced cell-mediated immunity as it also occurred in B-cell-deficient mice. Enolase immunization stimulated a predominant T-helper-1 (Th1) cytokine pattern in splenic cells and induced production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) by purified CD4+ T cells. However, despite the elevation of immunogenicity, recombinant enolase induced only a modest protection against disseminated candidiasis, suggesting a form of protection likely attributable to the induction of a Th1 cell-mediated immune response.

Dendritic cells transport conidia and hyphae of Aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate Th responses to the fungus.

Aspergilli are respiratory pathogens and pulmonary infections are usually acquired through the inhalation of conidia, able to reach small airways and the alveolar space where the impaired host defense mechanisms allow hyphal germination and subsequent tissue invasion. The invasive pulmonary aspergillosis is the most common manifestation of Aspergillus fumigatus infection in immunocompromised patients and is characterized by hyphal invasion and destruction of pulmonary tissue. A Th1/Th2 dysregulation and a switch to a Th2 immune response may contribute to the development and unfavorable outcome of invasive pulmonary aspergillosis. Dendritic cells (DC) have a primary role in surveillance for pathogens at the mucosal surfaces and are recognized as the initiators of immune responses to them. In the present study, we assessed the functional activity of pulmonary DC in response to A. fumigatus conidia and hyphae, both in vitro and in vivo. We analyzed mechanisms and receptors for phagocytosis by DC as well as DC migration, maturation, and Th priming in vivo upon exposure to either form of the fungus. We found a remarkable functional plasticity of DC in response to the different forms of the fungus, as pulmonary DC were able to: 1) internalize conidia and hyphae of A. fumigatus through distinct phagocytic mechanisms and recognition receptors; 2) discriminate between the different forms in terms of cytokine production; 3) undergo functional maturation upon migration to the draining lymph nodes and spleens; and 4) instruct local and peripheral Th cell reactivity to the fungus.

B7/CD28-dependent CD4+CD25+ regulatory T cells are essential components of the memory-protective immunity to Candida albicans.

Protective immunity to the fungus Candida albicans is mediated by Ag-specific Th1 cells. Paradoxically, some Th2 cytokines are required for the maintenance of Th1-mediated immune resistance to the fungus. Therefore, in addition to the Th1/Th2 balance, other mechanisms seem to be involved in the regulation of Th1 immunity to the fungus. Here we show that CD4(+)CD25(+) T cells, negatively regulating antifungal Th1 reactivity, are generated in mice with candidiasis. CD4(+)CD25(+) T cells were not generated in B7-2- or CD28-deficient mice or in condition of IL-10 signaling deficiency. Accordingly, although capable of efficiently restricting the fungal growth, these mice experienced inflammatory pathology and were incapable of resistance to reinfection. CD4(+)CD25(+) T cells poorly proliferated in vitro; were highly enriched for cells producing IL-4, IL-10, and TGF-beta; and required IL-10-producing, Candida hypha-activated dendritic cells for generation. Adoptive transfer of CD4(+)CD25(+) T cells or IL-10-producing dendritic cells restored resistance to reinfection and decreased inflammation in B7-2-deficient mice. These results show that oral tolerance induced by Candida hyphae is required for the occurrence of long-lasting protective immunity after yeast priming. The implication is that preventing reactivation rather than favoring sterilizing immunity to ubiquitous fungal pathogens may represent the ultimate expectation of vaccine-based strategies.

Dendritic cells pulsed with fungal RNA induce protective immunity to Candida albicans in hematopoietic transplantation.

Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-gamma-producing CD4(+) donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.

Protection of killer antiidiotypic antibodies against early invasive aspergillosis in a murine model of allogeneic T-cell-depleted bone marrow transplantation.

Antiidiotypic monoclonal antibodies (MAbs) representing the internal image of a yeast killer toxin (KT) have therapeutic potential against several fungal infections. The efficacy of KT MAbs against Aspergillus fumigatus was investigated in a mouse model of T-cell-depleted allogeneic bone marrow transplantation (BMT) with invasive pulmonary aspergillosis. Mice were highly susceptible to infection at 3 days post-BMT, when profound neutropenia was observed both in the periphery and in the lungs. Treatment with KT MAbs protected the mice from infection, as judged by the long-term survival and decreased pathology associated with inhibition of fungal growth and hyphal development in the lungs. In vitro, similar to polymorphonuclear neutrophils, KT MAbs significantly inhibited the hyphal development and metabolic activity of germinated Aspergillus conidia. These results indicate that mimicking the action of neutrophils could be a strategy through which KT MAbs exert therapeutic efficacy in A. fumigatus infections.

A role for antibodies in the generation of memory antifungal immunity.

Protective immunity to Candida albicans and Aspergillus fumigatus is mediated by antigen-specific Th1 cells. To define the role of B cells and antibodies in the generation of antifungal immune resistance, B cell-deficient (mu MT) mice were assessed for immune resistance to primary and secondary infections with both fungi. The results showed that, although passive administration of antibodies increased the fungal clearance, the innate and Th1-mediated resistance to the primary and secondary infections were both heightened in mu MT mice with candidiasis and aspergillosis. However, although capable of efficiently restricting the fungal growth, mu MT mice did not survive the re-infection with C. albicans, and this was concurrent with the failure to generate IL-10-producing dendritic cells and regulatory CD4(+)CD25(+) T cells. Antifungal opsonizing antibodies restored IL-10 production by dendritic cells from mu MT mice, a finding suggesting that the availability of opsonizing antibodies may condition the nature of the dendritic cell interaction with fungi, possibly impacting on the development of long-lasting antifungal immunity.

CD80+Gr-1+ myeloid cells inhibit development of antifungal Th1 immunity in mice with candidiasis.

To find out whether polymorphonuclear neutrophils (PMN), abundantly recruited in disseminated Candida albicans infection, could directly affect the activation of Th cells we addressed the issues as to whether murine PMN, like their human counterparts, express costimulatory molecules and the functional consequence of this expression in terms of antifungal immune resistance. To this purpose, we assessed 1) the expression of CD80 (B7-1) and CD86 (B7-2) molecules on peripheral, splenic, and inflammatory murine Gr-1+ PMN; 2) its modulation upon interaction with C. albicans in vitro, in vivo, and in human PMN; 3) the effect of Candida exposure on the ability of murine PMN to affect CD4+ Th1 cell proliferation and cytokine production; and 4) the mechanism responsible for this effect. Murine PMN constitutively expressed CD80 molecules on both the surface and intracellularly; however, in both murine and human PMN, CD80 expression was differentially modulated upon interaction with Candida yeasts or hyphae in vitro as well as in infected mice. The expression of the CD86 molecule was neither constitutive nor inducible upon exposure to the fungus. In vitro, Gr-1+ PMN were found to inhibit the activation of IFN-gamma-producing CD4+ T cells and to induce apoptosis through a CD80/CD28-dependent mechanism. A population of CD80+Gr-1+ myeloid cells was found to be expanded in conventional as well as in bone marrow-transplanted mice with disseminated candidiasis, but its depletion increased the IFN-gamma-mediated antifungal resistance. These data indicate that alternatively activated PMN expressing CD80 may adversely affect Th1-dependent resistance in fungal infections.